Characteristics Of Nitric Oxide Synthase Research Articles

Demonstration of nitric oxide synthase activity in crustacean hemocytes and anti-microbial activity of hemocyte-derived nitric oxide

We determined the biochemical characteristics of nitric oxide synthase (NOS) in hemocytes of the crayfish Procambarus clarkii and investigated the roles of hemocyte-derived NO in host defense. Biochemical analysis indicated the presence of a Ca 2+-independent NOS activity, which was elevated by lipopolysaccharide (LPS) treatment. When bacteria ( Staphylococcus aureus) and hemocytes were co-incubated, adhesion of bacteria to hemocytes was observed. NO donor sodium nitroprusside (SNP) significantly increased the numbers of hemocytes to which bacteria adhered. Similarly, LPS elicited bacterial adhesion and the LPS-induced adhesion was prevented by NOS inhibitor N G-monomethyl- l-arginine ( l-NMMA). Finally, plate count assay demonstrated that addition of LPS to the hemocytes/bacteria co-incubation resulted in a significant decrease in bacterial colony forming unit (CFU), and that l-NMMA reversed the decreasing effect of LPS on CFU. The combined results demonstrate the presence of a Ca 2+-independent LPS-inducible NOS activity in crayfish hemocytes and suggest that hemocyte-derived NO is involved in promoting bacterial adhesion to hemocytes and enhancing bactericidal activity of hemocytes.

Characterization of nitric oxide synthase in the rat parotid gland

Nitric oxide (NO) acts as an inter- and intracellular signalling molecule of various cells such as vascular endothelium, macrophages, and neurones. NO is produced by nitric oxide synthase (NOS) from l-arginine. Here the characteristics of NOS in the rat parotid gland were investigated. Approximately 74% of total activity of NOS was present in the cytosolic fraction. For full activation of the NOS in the cytosolic fraction, tetrahydroxybiopterin, NADPH, Ca 2+ and calmodulin were needed as cofactors, because the activity was clearly reduced in the absence of tetrahydroxybiopterin, NADPH, or Ca 2+, or in the absence of calmodulin and presence of trifluoperazine, a calmodulin antagonist, in the reaction mixture. The partially purified NOS activity was completely abolished in the absence of calmodulin or Ca 2+, and activated by them in a dose-dependent manner; EC 50 for calmodulin and Ca 2+ were 10 and 340 nM, respectively. The K m for l-arginine was 1.57 μM. Immunoblot analysis revealed that a 165-kDa protein band in the rat parotid gland cytosolic fraction cross-reacted with a rabbit polyclonal antibody against human brain NOS. These results suggest that NOS of the rat parotid gland is a neuronal isoform and that its activity is regulated by physiological concentrations of calmodulin and Ca 2+.

Location and Characteristics of Nitric Oxide Synthase in Sheep Spinal Cord and Its Interaction with α2-Adrenergic and Cholinergic Antinociception

Nitric oxide synthase is located in the spinal cord dorsal horn and intermediolateral cell column, where it may modulate sensory and sympathetic neuronal activity. However, the biochemical characteristics of this enzyme have not been examined in these different areas in the spinal cord. Although alpha(2)-adrenergic agonists, muscarinic agonists, and nitric oxide may interact in the spinal cord to produce antinociception, these interactions have not been characterized. Sheep spinal cord tissue was homogenized ad centrifuged at high sped to separate soluble and membrane-bound fractions. Nitric oxide synthase activity was determined by conversion of [(14)C]-L-arginine to [(14)C]-L-citrulline and its kinetic characteristics, dependency on cofactors, and sensitivity to inhibitors determined. Sheep spinal cord was stained for nicotinamide adenine dinucleotide phosphate diaphorase as a marker for nitric oxide synthase. Antinociception to a mechanical stimulus from intrathecal clonidine alone and with neostigmine was determined and the effects of L-arginine and n-methyl-L-arginine were determined. More than 85% of nitric oxide synthase activity was present in the soluble form and its kinetic, cofactor, and antagonist properties were similar to those of the neuronal isoform of nitric oxide synthase. Biochemical and histochemical studies localized nitric oxide synthase to the superficial dorsal horn and the intermediolateral cell column. Clonidine antinociception was enhanced by L-arginine and neostigmine, but not by D-arginine. Neostigmine's enhancement of clonidine antinociception was blocked by n-methyl-L-arginine. These results confirm those of previous studies demonstrating localization of nitric oxide synthase to superficial dorsal horn and intermediolateral cell column of mammalian spinal cord, and suggesting its identity as the neuronal isoform. Spinal alpha(2)-adrenergic agonist antinociception may be partly dependent on cholinergic and nitric oxide mechanisms.

Estrogen increases nitric oxide synthase activity in the uterus of nonpregnant sheep

OBJECTIVE: The aim of this study was to characterize nitric oxide synthase activity in endometrium and myometrium of nonpregnant sheep and to determine whether estrogen administration affects uterine nitric oxide synthase activity. STUDY DESIGN: Nonpregnant sheep were castrated during synchronized estrus and 4 days after surgery were treated with 100 μg/day of 17β-estradiol for 3 days. Nitric oxide synthase activity was measured by the citrulline conversion assay. RESULTS: Citrulline generation found in soluble and particulate fractions had all the characteristics of nitric oxide synthase, namely, it was strictly dependent on reduced nicotinamide adenine dinucleotide phosphate and enhanced by flavin nucleotides and tetrahydrobiopterin. Estrogen administration significantly increased Ca ++-dependent nitric oxide synthase activity in myometrium but not in endometrium. The effect was more pronounced in the membrane-associated enzyme activity (approximately fivefold). Estrogen treatment increased myometrial nitric oxide synthase activity from 9.0 ± 2.4 to 20.0 ± 3.7 pmol/mg of protein per 30 minutes in the soluble fraction and from 12.0 ± 5.1 to 62.0 ± 13.1 pmol/mg of protein per 30 minutes in the particulate fraction (mean ± SEM, p < 0.05 by t test). The increase in nitric oxide synthase activity was not mediated by an increase in tetrahydrobiopterin availability, as shown to be the case in macrophages. CONCLUSION: These data show that in the nonpregnant sheep uterus > 90% of the nitric oxide synthase activity found in myometrium is Ca ++ dependent and is up-regulated by estrogen in a tissue-specific manner.

Kinetic characteristics of nitric oxide synthase from rat brain

Kinetic characteristics of nitric oxide synthase from rat brain

Kinetic characteristics of nitric oxide synthase from rat brain

The relationship between the rate of synthesis of nitric oxide (NO) and guanylate cyclase stimulation was used to characterize the kinetics of the NO synthase from rat forebrain and of some inhibitors of this enzyme. The NO synthase had an absolute requirement for L-arginine and NADPH and did not require any other cofactors. The enzyme had a Vmax. of 42 pmol of NO formed.min-1.mg of protein-1 and a Km for L-arginine of 8.4 microM. Three analogues of L-arginine, namely NG-monomethyl-L-arginine, NG-nitro-L-arginine and NG-iminoethyl-L-ornithine inhibited the brain NO synthase. All three compounds were competitive inhibitors of the enzyme with Ki values of 0.7, 0.4 and 1.2 microM respectively.